Control of Ostrinia

ABSTRACT

A method to combat or control Ostrinia nubilalis by contacting such insects with a CryIB protein or a combination of a CryIB protein and a CryIAb or CryIAc protein.

This application is a continuation of application Ser. No. 08/164,781, filed Dec. 10, 1993 now abandoned, which is a continuation of Ser. No. 07/938,36 filed Aug. 31, 1992, now abandoned.

This invention relates to a method to control or combat Ostrinia, particularly Ostrinia nubilalis (Lepidoptera, Pyralidae) or the European corn borer, using a Bacillus thuringiensis ("Bt") cryIB gene or CryIB protein or using the cryIB gene and the cryIAb or cryIAc gene or their respective proteins. This invention also relates to a method to protect crops, particularly corn, against Ostrinia.

This invention further relates to the use of microorganisms, especially plant-associated microorganisms, preferably Clavibacter xyli, and to the use of plants, especially monocotyledonous plants, particularly corn (maize, Zea mays), stably transformed with the cryIB gene alone or with both the cryIB gene and cryIAb or cryIAc gene to control or combat Ostrinia such as O. nubilalis.

This invention still further relates to the use of insecticidal formulations containing the CryIB protein or both the CryIB protein and the CryIAb or CryIAc protein to protect plants from Ostrinia.

This invention also relates to a plant, especially a monocot, particularly a cereal plant, quite particularly corn, infestable by O. nubilalis and transformed with an expressible cryIB gene or with both an expressible cryIB gene and a cryIAb or cryIAc gene, to combat or control Ostrinia.

BACKGROUND OF THE INVENTION Bacillus Thuringiensis

Bacillus thuringiensis ("Bt") is a gram-positive soil bacterium, which produces endogenous crystalline inclusions upon sporulation. Early in this century, these bacteria were found to be insecticidal (Berliner, 1915). Some years later, their insecticidal activity was found to reside in the proteins present in their crystals, hereinafter referred to as "insecticidal crystal proteins" or "ICPs". Since then, the Bt strains, spores, crystals and ICPs have been used as biological insecticides in commercial formulations.

The limited spectrum of these insecticidal proteins allows any naturally occurring predators of the target insects to survive. The continued presence of these predators prevents further outbreaks of the insects. Furthermore, these Bt proteins have the advantage that they are rapidly degradable and that no stable residues accumulate in the environment.

Cry Proteins and Cry Genes

The specificity of the environmentally safe Bt insecticides has provoked a search for new Bt strains, producing toxins against other insect pests. Insecticidal Bt strains toxic to lepidopteran, coleopteran and dipteran insects have been found (H ofte and Whiteley, 1989). Although considerable homology can be found between genes that encode various ICPs toxic to one particular insect class, the sensitivity of specific insects to related Bt gene products is often very different. For instance, Chambers et al (1991) described a large difference in activity of the CryIF protein against Heliothis virescens and Heliothis zea (50% lethal concentrations of respectively 0.31 and >57 ng protoxin/mm² diet).

The Bt insecticidal crystal (Cry) proteins have been divided into five classes, according to their structural similarities and insecticidal spectra (H ofte and Whiteley, 1989): CryI proteins are toxic to Lepidoptera, CryII proteins are toxic to Diptera and Lepidoptera, CryIII proteins are toxic to Coleoptera and CryIV proteins are toxic to Diptera. A general cytolytic protein (cytA) is classified as a fifth toxic protein, but it has no specific insecticidal activity. The Bt genes coding for the insecticidal Cry proteins (cry genes) show strong homology in some conserved domains. These insecticidal Bt genes are mostly found on large conjugative plasmids, which may explain their observed mobility among Bt strains. One strain can contain several cry genes, and one gene can be found in several strains (H ofte and Whiteley, 1989).

Typically, cryI genes encode proteins with a molecular weight of 130 to 140 kD (hereinafter referred to as the "protoxins"), and upon ingestion by a sensitive insect, the protoxins are processed to smaller proteins (hereinafter referred to as the "toxins") having a molecular weight of 60 to 70 kD. The cryII and cryIII genes encode protoxins with a molecular weight of about 70 kD (except the cryIIIC gene which encodes a protoxin of 129 kD according to PCT publication WO 90/09445). The CryIV genes encode protoxins of either of these molecular weight types. The CryI protoxins constitute the largest group of protoxins, which are found in typical bipyramidal crystals.

The cry genes have been used to transform bacteria (e.g., Obukowicz et al, 1986; Stock et al, 1990) and plants (e.g., Vaeck et al, 1987) in order to provide resistance against insect pests. Adequate expression in plants was only obtained when the plants were transformed with a truncated Bt gene (e.g., Vaeck et al, 1987; Fischhoff et al, 1987; Barton et al, 1987).

The cryIB gene has been described in European patent publication ("EP") 408 403 and by Brizzard and Whiteley (1988). It encodes a 137 kD protoxin and a 66 kD toxin. The CryIB toxin has been shown to be insecticidal to insects like Pieris brassicae, Plutella xylostella, Spodoptera littoralis and Spodoptera exigua (Ferr e et al, 1991; Visser et al, 1988).

The CryIAa (Gawron-Burke and Baum, 1991), CryIAb, CryIAc (Macintosh et al, 1990) and CryIF gene products (Chambers et al, 1991) have been described as toxic to O. nubilalis. Moreover, Peferoen (1991) has described the insecticidal activity of the following ICPs against various insects, including O. nubilalis: CryIAa, CryIAb, CryIAc, CryIB, CryID, CryIC and CryIE, and PCT publication WO 92/09696 also has described the insecticidal activity of the cryIAb and cryIB genes against O. nubilalis.

PCT publication 90/15139 has described the prevention of insect resistance development with various combinations of Bt genes, such as the cryIAb and cryIB genes (the Bt 2 and Bt 14 genes), against Pieris brassicae, Plutella xylostella, and Phthorimaea operculella.

Mode of Action of the CryI Proteins

The ICPs owe their specificity to the presence of specific receptor sites in the midgut brush border membranes of sensitive insects. In vivo, the crystals are solubilized in the alkaline environment of the midgut, and the released protoxins are processed by proteases to yield smaller protease-resistant toxins which bind to, and cause swelling of, the midgut cells (Gill et al, 1992). The C-terminal part of the CryI-type protoxin is probably involved in the formation of its crystal structure, but is thought not to be important in its mode of action (H ofte and Whiteley, 1989). Electrophysiological evidence (Harvey et al, 1983) and biochemical evidence (Knowles and Ellar, 1987) suggest that the toxins generate pores in the midgut brush border cell membranes, thus disturbing the osmotic balance. The intoxicated insects quickly stop feeding and eventually die. The high affinity binding of the toxins has been correlated with their toxicity (Van Rie et al, 1989).

Ostrinia Nubilalis

The European corn borer is a very serious and persistant pest for corn (Davidson and Lyon, 1987; Hudon et al, 1987). The larvae of this insect initially feed on leaf tissue and later enter the stalks, burrowing downwards as the season progresses. O. nubilalis is estimated to be the most important corn pest in Europe and the second most important in the USA. Damage caused by O. nubilalis in the USA is estimated to be over 400 million dollars (U.S.) a year. Estimates for O. nubilalis spraying amount in France to 25 million dollars (U.S.) a year. Up to now, hazardous chemical insecticides have mostly been used to combat this insect. The European corn borer is remarkably polyphagous (Hudon et al, 1987) and has been found to attack other important crops such as wheat, cotton, potato, tomato, beet, oat and soybean plants (Davidson and Lyon, 1987; Hudon et al, 1987).

SUMMARY OF THE INVENTION

In accordance with this invention, a method is provided to combat and/or control insects of the species Ostrinia, particularly Ostrinia nubilalis (the European corn borer), by the step of contacting these insects with: a) the CryIB protein or an equivalent thereof; or b) both i) the CryIB protein and ii) the CryIAb or the CryIAc, preferably the CryIAb, protein or their equivalents.

Also in accordance with this invention, the contacting step can be carried out with an insecticidal composition comprising: the CryIB protein or its equivalent or both the CryIB protein and the CryIAb or CryIAc protein or their equivalents in pure form; or Bt crystals containing these protein(s) or their equivalents; or crystal-spore mixtures of naturally occurring Bt bacteria containing the cryIB gene or its equivalent or both the cryIB gene and the cryIAb or cryIAc gene or their equivalents; or crystal-spore mixtures of Bt bacteria transformed with an expressible cryIB gene or its equivalent or with both an expressible cryIB gene and an expressible cryIAb or cryIAc gene or their equivalents.

Further in accordance with this invention, the contacting step can be carried out with a microorganism, preferably a plant-associated microorganism, especially an endophytic microorganism, particularly Clavibacter xyli, transformed with an expressible cryIB gene or its equivalent or with both an expressible cryIB gene and an expressible cryIAb or cryIAc gene or their equivalents, so as to inoculate plants or parts thereof, such as seeds, so that they become resistant to attack by Ostrinia.

Furthermore, the contacting of the insects can be with a plant, especially a monocotyledonous plant, particularly a cereal plant, quite particularly corn, stably transformed with an expressible cryIB gene or its equivalent or with both an expressible cryIAb or cryIAc gene and an expressible cryIB gene or their equivalents, so that the transformed plant expresses the CryIB protein or its equivalent or a combination of the CryIB and CryIAb or CryIAc proteins or their equivalents in insecticidally effective amounts.

Moreover, a plant, especially a monocotyledonous plant, particularly a cereal plant, quite particularly a corn plant, infested by Ostrinia, is protected from this insect by having been stably transformed with the cryIB gene or its equivalent or with both the cryIB gene and the cryIAb or cryIAc gene or their equivalents.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the result of toxicity assays which were conducted by feeding the European corn borer, Ostrinia nubilalis, an artificial diet containing the purified CryIB toxin and which surprisingly showed that this protein was toxic to the European corn borer (see Example 1). Furthermore, the CryIB toxin was found to bind non-competitively to the midgut membranes of Ostrinia when compared with other Cry toxins which are insecticidally active against this insect as shown in Example 2. Therefore, this active Bt protein can be used to provide maximum protection against this important pest and can prevent or reduce the development of insect resistance to Bt insecticidal formulations in the field.

The "CryIB protein" of this invention encompasses the full length protein (protoxin) encoded by the cryIB gene and having the amino acid sequence shown in SEQ ID No. 1 of the Sequence Listing and any protein that is substantially the same as the CryIB protoxin of SEQ ID No. 1, as well as any insecticidally active fragment thereof, such as the CryIB toxin. An example of substantially the same protein as the protoxin of SEQ ID No. 1 is the naturally occurring CryIB protoxin described by Brizzard and Whiteley (1988). The "CryIB protein" of this invention includes proteins in which some amino acids of the protoxin of SEQ ID No. 1 are deleted, added or replaced by others without significantly changing the insecticidal activity, particularly against O. nubilalis, for example the modified CryIB protoxin described in EP 408 403. "CryIB toxin" as used herein, means the smallest insecticidally active fragment of the CryIB protoxin, extending from amino acid 145 to amino acid 636 in SEQ ID No. 1 In this regard, "insecticidally active fragment of the CryIB protoxin" as used herein, means any part of the CryIB protoxin having insecticidal activity, preferably its toxin.

In this invention, "cryIB gene" encompasses the gene with the DNA sequence shown in SEQ ID No. 1 of the Sequence Listing or any mutant, synthetic or modified gene encoding a CryIB protein, such as the modified gene described in EP 408 403. Modifications to the gene can include: 1) the replacement of some codons with others coding for the same or for other amino acids, preferably with codons that code for the same amino acids; 2) deleting or adding some codons; and 3) reciprocal recombination as described by Ge et al (1991); provided that such modifications do not substantially alter the properties, especially the insecticidal properties, particularly against O. nubilalis, of the encoded CryIB protein. It is evident that the definition of the cryIB gene comprises any modified gene designed to provide higher expression levels of a CryIB protein in plants. One particularly preferred modified gene is the naturally occurring cryIB gene described by Brizzard and Whiteley (1988), wherein only two nucleotides are different from SEQ ID No. 1: in the Brizzard and Whiteley sequence, a T is replaced by a C at position 311, and a C is replaced by a T at position 633. Only the latter change in Brizzard and Whiteley leads to a different amino acid: a His codon is changed to a Tyr codon. "Insecticidally active fragment of the cryIB gene", as used herein, means any truncated gene encoding an insecticidally active fragment of the CryIB protein, like the gene fragment encoding the CryIB toxin.

In accordance with this invention, a cryIB gene can be isolated from a Bt strain, for example Bt. entomocidus HD-110. This strain is publicly available from the Agricultural Research Culture Collection, Northern Regional Research Laboratory, U.S. Dept. of Agriculture, Peoria, Ill. 61604, USA ("NRRU"). The isolation and cloning of the cryIB gene, as well as its modification, are described in EP 408 403. The gene has an open reading frame (ORF) of 3684 bp, encoding a 137 kD protoxin and 66 kD and 55 kD protease-activated fragments. The nucleotide sequence and the corresponding amino acid sequence are shown in SEQ ID No. 1. An insecticidally active cryIB gene fragment also can be constructed as described in EP 408 403. For this purpose, a BclI site has been identified downstream of the coding sequence encoding the CryIB toxin.

Similarly, the "CryIAb protein" of this invention encompasses a protoxin with the amino acid sequence disclosed in EP 193 259 and shown in SEQ ID No. 2 in the Sequence Listing, any protein that is substantially the same as the CryIAb protoxin of SEQ ID No. 2, and any insecticidally active fragment thereof, such as the CryIAb toxin. The CryIAb protein includes: naturally occurring variants with substantially the same insecticidal activity, particularly against O. nubilalis, such as the CryIAb protoxin described by H ofte and Whiteley (1989) and in EP 224 331 and the CryIAb protoxin described by Fischhoff et al (1987); and any CryIAb protoxin encoded by a modified or synthetic Bt gene but with substantially the same insecticidal activity as the protoxin of SEQ ID No. 2, as described, for example, in PCT publication WO 91/16432 and in European patent applications ("EPA") 91402920 2 and 92400820.4. "CryIAb toxin" as used herein, is the protein containing amino acids 29 to 601 of the amino acid sequence shown in SEQ ID No. 2 in the Sequence Listing. "Insecticidally active fragment of the CryIAb protein", as used herein, means any fragment of the CryIAb protoxin having insecticidal activity, preferably the cryIAb toxin.

Similarly, the "cryIAb gene" of this invention encompasses the gene with the DNA sequence shown in SEQ ID No. 2 or any mutant, synthetic or modified gene encoding a CryIAb protein. Naturally occurring cryIAb genes with minor differences include the gene described in EP 224 331 and the genes listed by H ofte and Whiteley (1989). Modifications, as described above for the cryIB gene, can also be introduced into the cryIAb gene, provided that such modifications do not substantially alter the insecticidal properties, particularly against O. nubilalis, of the encoded CryIAb protein. The isolation and cloning of a cryIAb gene is described in EP 193 259 and by H ofte et al (1986). The gene contains an ORF of 3464 bp, encoding a protoxin of 131 kD and a toxin of 60 kD. The gene can be isolated from the Bt subsp. thuringiensis berliner 1715 strain (H ofte et al, 1986) or from the Bt HD-1 kurstaki strain which is publicly available from the N.R.R.L.

Likewise, the "CryIAc protein" of this invention encompasses a protoxin with the amino acid sequence shown in SEQ ID No. 3 of the Sequence Listing, any protein that is substantially the same as the CryIAc protoxin of SEQ ID No. 3 and any insecticidally active fragment thereof, such as the CryIAc toxin described by Dardenne et al (1990).

Likewise, the "cryIAc gene" of this invention encompasses the gene described by Adang et al (1985) with the DNA sequence as shown in SEQ ID No. 3 of the Sequence listing or any mutant, synthetic or modified gene, encoding a CryIAc protein. Variants of the cryIAc gene include: the modified or synthetic cryIAc genes described in EP 358 962; and the naturally occurring cryIAc gene described by Dardenne et al (1990), EP 367 474, and PCT publication WO 90/03434 which is a preferred variant differing from the cryIAc DNA sequence of SEQ ID No. 3 by 10 nucleotides in the gene part encoding the toxin (one nucleotide triplet also being deleted in this part, resulting in 3 different amino acids in the toxin and one deleted amino acid). The cryIAc gene can be isolated from the Bt subsp. thuringiensis HD-73 strain, publicly available from the NRRL.

In accordance with this invention, one can combat or control Ostrinia species, particularly the European corn borer, by contacting this insect: a) with the CryIB protein or b) with a combination of the CryIB protein and the CryIAb protein or a combination of the CryIB protein and the CryIAc protein, preferably the combination of the CryIB and CryIAb proteins. Such combinations of proteins encompass combinations of the full length protoxins and/or insecticidally active fragments of such protoxins, achieved for example by co-expression of the corresponding genes and gene fragments in a cell or by expression of a modified gene encoding insecticidally active fragments of both proteins. By "combat" is meant treating plants in a field in such a way as to destroy the Ostrinia (e.g. European corn borers) that are attacking or that would attack the plants such as when a sudden increase in its population would occur; by "control" is meant treating plants in a field in such a way as to limit the Ostrinia's damage to the plants such as when relatively small numbers of insects are constantly present in the field without causing major damage to the plants; and by "contacting" is meant ensuring that the CryIB protein or a combination of the CryIB and CryIAb or CryIAc proteins is present in a field of plants that is infested, or can be infested, by Ostrinia so that the protein(s) can become ingested by the insects, for example by transforming either the plants, plant-associated microorganisms or other microorganisms, or by applying to the field insecticidal formulations containing the CryIB protein or the combination of the CryIB protein and the CryIAb or CryIAc protein.

Contacting Ostrinia with the CryIB protein or a mixture thereof with the CryIAb or CryIAc protein in accordance with this invention can be carried out directly by using an insecticidal composition comprising the CryIB protein or both the CryIB and CryIAb or CryIAc proteins in the form of purified proteins, in the form of Bt strains or their crystals, or in the form of Bt crystal-spore mixtures. By "purified proteins" is meant the CryIB, CryIAb and/or CryIAc proteins purified from their crystal proteins, from transformed microorganisms or from transformed plant cells by methods well known in the art (e.g., as described in EP 193 259). In this regard, such a contacting step can be carried out with naturally occurring or genetically engineered Bt strains, preferably the Bt subsp. entomocidus HD-110, Bt subsp. thuringiensis HD-2 or Bt subsp. thuringiensis 4412 strain (H ofte et al, 1986; H ofte and Whiteley, 1989), containing the cryIB gene or both the cryIB and cryIAb genes. For contacting the insects with both the CryIB and CryIAb protoxins, the Bt subsp. thuringiensis HD-2 strain is preferred, since it has been found to comprise both the cryIB and cryIAb genes (Brizzard et al, 1991).

An insecticidal, particularly an anti-Ostrinia, composition comprising the CryIB protein or the CryIB and the CryIAb or CryIAc proteins can be formulated in a conventional manner, together with suitable carriers, diluents, emulsifiers and/or dispersants known in the art. Also, well known methods for stabilizing Cry proteins in the field can be used, such as by delivering the proteins to the field in killed and stabilized microorganisms, or targeting the proteins, synthesized by plants or microorganisms transformed with the cryIB gene or the cryIAb or cryIAc and the cryIB genes to certain intra- or extracellular sites where a higher stability of the proteins can be obtained.

The CryIB protein or the CryIB and CryIAb or CryIAc proteins or killed and stabilized cells of microorganisms containing such proteins can be formulated in insecticidal compositions in a variety of ways, using any number of conventional additives, wet or dry, depending upon the particular use. Additives can include wetting agents, detergents, stabilizers, adhering agents, spreading agents and extenders. Examples of such compositions include pastes, dusting powders, wettable powders, granules, baits and aerosol sprays. Other Bt proteins or killed and stabilized cells of microorganisms containing such proteins and other insecticides, as well as fungicides, biocides, herbicides and fertilizers, can be employed along with the CryIB protein or the CryIB and the CryIAb or CryIAc proteins or killed and stabilized cells containing such proteins to provide additional advantages or benefits. Such an insecticidal composition can be prepared in a conventional manner, and the amount of the CryIB protein or the CryIB and the CryIAb or CryIac proteins employed will depend upon a variety of factors, such as the composition used, the type of area to which the composition is to be applied, and the prevailing weather conditions, but generally the concentration of such proteins will be at least about 0.1% by weight of the formulation, more often from about 0.15% to about 0.8% by weight percent of the formulation.

The cryIB gene or the cryIB and the cryIAb or cryIAc genes can, if desired, also be used with their native 5' and 3' signal sequences, to transform microorganisms such as Bt strains, in order to control Ostrinia, particularly O. nubilalis. Of course, other microorganisms can be transformed, such as phages and other viruses, bacteria, fungi and yeasts. Such transformations can be carried out in a conventional manner, preferably by using conventional electroporation methods as described in PCT publication WO 90/06999 or other methods as described by Lereclus et al (1992). To obtain expression in microorganisms other than Bt, such cry genes will have to contain the necessary signal sequences to provide proper expression in such other microorganisms. The BtPGSI387 strain (PCT publication WO 90/06999) is particularly suited for transformation with such cry genes, since this strain is easily fermented by conventional methods (Dulmage, 1981) to provide high yields of cells. The so-transformed microorganism can then be used to produce the CryIB protein or the CryIB and the CryIAb or CryIAc proteins, which could then be formulated for protecting plants from Ostrinia.

Contacting Ostrinia, particularly the European corn borer, with the CryIB protein or mixtures thereof in accordance with this invention can also be carried out indirectly, by ensuring that the CryIB protein or the CryIB and the CryIAb or CryIAc proteins are biologically produced at appropriate places by microorganisms or plants expressing the cryIB gene or the cryIAb or cryIAc and the cryIB genes. This can be achieved by inoculating plants or parts of plants, like seeds, with plant-associated microorganisms, transformed with the cryIB gene or the cryIB and the cryIAb or cryIAc genes. By "inoculating" is meant contacting or coating a plant or part of a plant with the microorganisms such that they remain associated with the plant or plant parts. Plant-associated microorganisms, which can be used, include the plant-colonizing (epiphytic) microorganisms like the Pseudomonas bacteria and endophytic plant-colonizing microorganisms like Clavibacter xyli. Transformation of Clavibacter xyli subsp. cynodontis with the cryIB gene or the cryIB and the cryIAb or cryIAc genes can be carried out as described by Turner et al (1991), and these genes are preferably under the control of their original Bt promoter or any other Bt promoter and are flanked by suitable 3' transcription termination signals like the lambda tR1 transcription terminator sequence (Turner et al, 1991). Stably transforming plants with the cryIB gene or with a combination of the cryIAb or cryIAc and the cryIB genes in accordance with this invention also renders the plants and their progeny resistant to Ostrinia.

In order to express the cryIB gene or the cryIB and the cryIAb or cryIAc genes in microorganisms and plants, suitable restriction sites can be introduced, flanking the gene(s). This can be done by site-directed mutagenesis (Stanssens et al, 1989).

In order to obtain enhanced expression in plants, it may be preferred to modify the cryIB, cryIAb and/or cryIAc genes as described: in PCT publication WO 91/16432, EPA 91402920.2 and 92400820.4 and by Perlak et al (1991) and Murray et al (1991). A particularly preferred modification to the cryIB gene involves changing the exceptional TTG start codon to the more common ATG start codon by site-directed mutagenesis (Stanssens et al, 1989) as described in EP 408 403.

A gene cassette, containing the cryIB gene or the cryIB and the cryIAb or cryIAc genes, can be constructed as described in EP 408 403 in order to express the gene(s) in E. coli and plants. In this regard, insecticidally effective part(s) of such gene(s) can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a stably transformed plant that is resistant to the European corn borer. In this regard, a disarmed Ti-plasmid, containing the insecticidally effective gene part(s), in Agrobacterium tumefaciens can be used to transform the plant cell using the procedures described, for example, in EP 116 718, EP 270 822, PCT publication WO 84/02913, Deblaere et al (1985), and Gould et al (1991). Preferred Ti-plasmid vectors contain the insecticidally effective gene part(s) between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform the plant cell, such as direct gene transfer (as in EP 233 247), pollen-mediated transformation (as in PCT publication WO 85/01856), plant RNA virus-mediated transformation (as in EP 067 553), or liposome- mediated transformation (as in US patent 4,536,475). Other methods described for transforming certain lines of corn (Fromm et al, 1990; Gordon-Kamm et al, 1990) and the more recently described method for transforming monocots generally (PCT publication WO 92/09696) also can be used.

The resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective gene part(s) in other varieties of the same or related plant species. The seeds, obtained from these plants, contain the respective gene part(s) as stable genomic inserts. Cells of the transformed plant can be cultured to produce the gene products for use in conventional insecticidal compositions.

Part(s) of the cryIB or the cryIB and the cryIAb or cryIAc gene(s), encoding insecticidally active fragment(s) of the CryIB or the CryIB and the CryIAb or CryIAC proteins, are inserted in a plant cell genome so that the inserted gene part(s) are downstream (e.g. 3') of, and under the control of, a promoter which can direct the expression of the gene part(s) in the plant cell; and upstream (e.g. 5') of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). Preferred promoters include: the strong constitutive 35S promoters of the cauliflower mosaic virus of isolates CM 1841 (Gardner et al, 1981), CabbB-S (Franck et al, 1980) and CabbB-JI (Hull and Howell, 1987); and the TRi' promoter and the TR2' promoter which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al, 1984). Alternative promoters are those which are selectively expressed in certain tissues or are inducible promoters (such as the promoter of the ribulose-1,5-bisphosphate carboxylase small subunit gene disclosed in EP 193 259). Preferred polyadenylation and transcript formation signals include those of the octopine synthase gene (Gielen et al, 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3' untranslated DNA sequences in transformed plant cells. For example, the cryIB gene or the cryIB and the cryIAb or cryIAc genes can be inserted into the pDEl10 or pDE108 vector, described in PCT patent publication WO 92/09696, under the control of suitable plant promoters and flanked by suitable 3' termination sites as described above. These vectors can be used to stably transform corn lines with these genes (e.g., as described in PCT publication WO 92/09696), thus rendering the corn lines resistant to attack by Ostrinia, such as the European corn borer.

To achieve co-expression of the cryIB and the cryIAb or cryIAc gene(s) in plants, it is preferred that two plants, each transformed with one of the cry genes, be crossed to obtain a progeny, containing both genes as described, for example, in EP 408 403. The resulting plants are well protected against Ostrinia nubilalis attack by the expression of both the CryIB and the CryIAb or CryIAc proteins in the plant cells. Gene cassettes for co-expression of the cryIB and cryIAb genes in plants are described in EP 408 403. For obtaining enhanced expression in monocots such as corn, the cryIAb or cryIAc and the cryIB genes are preferably modified as described in PCT publication WO 91/16432 and in EPA 91402920.2 and 92400820.4. These modified genes can be transferred to a monocot cell by electroporation as disclosed in PCT publication WO 92/09696 to achieve expression of the genes in monocots after regeneration of the monocot cell to a plant.

It is also preferred to provide the transformed plant cells with screenable or selectable marker genes. Suitable marker genes include the neogene (Reiss et al, 1984; EP 242 236), coding for kanamycin resistance. The transformed cells can be provided with a hybrid gene, containing the cry gene(s) and the marker gene under the control of the same promoter. This hybrid gene will be expressed in the transformed cells as a fusion protein (Vaeck et al, 1987). Also hybrid genes, comprising the active fragments of both the cryIB and the cryIAb or cryIAc genes, can be constructed as described by, for example, Ge et al (1991).

The following Examples illustrate the invention. In the Examples, all procedures for making and manipulating DNA are carried out by the standard procedures described by Sambrook et al (1989) Molecular Cloning--A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, N.Y., USA.

In the Examples, references are made to the following Figures and Sequence Listing.

FIG. 1 shows the binding of iodinated CryIAb toxin to brush border membrane vesicles of O. nubilalis. Membrane vesicles were incubated (30 min.) with iodinated CryIAb toxin in the presence of increasing concentrations of competitor: unlabeled CryIAb (*), CryIAc (∘) and CryIB (□) toxins. The CryIB toxins did not bind to the receptors occupied by the labeled CryIAb toxins, while the CryIAc and CryIAb toxins suppress binding of the labelled CryIAb toxins. Curves were predicted by the LIGAND computer program (Munson and Rodbard, 1980 ) . Each point is the mean of three independent experiments (three independently prepared batches of vesicles) .

SEQUENCE LISTING

SEQ ID No. 1 is the nucleotide sequence of the cryIB gene and the corresponding amino acid sequence of the CryIB protoxin as described in EP 408 403.

SEQ ID No. 2 is the nucleotide sequence of the cryIAb gene and the corresponding amino acid sequence of the CryIAb protoxin as described in EP 193 259.

SEQ ID No. 3 is the nucleotide sequence of the cryIAc gene and the corresponding amino acid sequence of the CryIAc protoxin.

EXAMPLE 1 Insecticidal Activity of the CryIB Toxin

The CryIB toxin of SEQ ID No. 1, obtained from Bacillus thuringiensis subsp. entomocidus HD-110, was found to be insecticidal to neonate Ostrinia nubilalis (European corn borer) larvae in bio-assays on artificial diet (diet according to Poitout et al, 1972).

Multiwell plates were filled with the artificial diet, and sample dilutions of different purified CryI toxins (50 μl) in bovine serum albumin-containing phosphate buffered saline ("PBS-BSA": 8 mM Na₂ HPO₄, 2 mM KH₂ PO₄, 150 mM NaCl and 0.1% BSA) were applied uniformly on the surface of the food and allowed to dry. Mortality was scored after 5 days. The toxicity data were analyzed by probit analysis (Finney, 1962).

As shown in Table I below, O. nubilalis is very sensitive to the CryIB toxin. The 50% lethal concentration value is much lower than most other CryI toxins and only the CryIAb toxin is more toxic. Also, the toxicity of the CryIB protoxin against O. nubilalis larvae was found to be comparable to that of the activated toxin, demonstrating that the proteolytical activation of the protoxin in the midgut did not interfere with toxicity.

                  TABLE I                                                          ______________________________________                                         LC.sub.50 values of solubilized purified CryI toxins to                        O. nubilalis (LC.sub.50 expressed in ng toxin/cm.sup.2 of                      ______________________________________                                         diet).                                                                         CryI Toxin                                                                             IAa    IAb    IAc   IB   IC    ID    IE                                LC.sub.50                                                                              1247   50     531   105  >1350 >1350 >1350                             ______________________________________                                    

Upon spraying of corn plants with the CryIB toxin, the plants are protected from O. nubilalis larvae, which immediately stop feeding upon spraying and do not cause any major damage to the plants.

EXAMPLE 2 Binding of the CryIB, CryIAb and CryIAc Toxins to O. nubilalis Midgut Membranes

Receptor binding assays were conducted to compare the binding of the CryIB toxin with that of other CryI toxins. These tests were conducted on Ostrinia midgut brush border membrane vesicles, prepared as described by Wolfersberger et al (1987), using radioligand competition binding experiments as described by Van Rie et al (1989).

As shown in FIG. 1, the CryIAb and CryIAc toxins bind to the same receptor sites in the brush border membranes. However, no suppression of CryIAb binding is obtained when adding the CryIB toxin to the assay, indicating that the CryIB toxin binds to a different receptor.

Furthermore, immunocytochemical assays, using polyclonal antibodies against the CryIB toxin, showed accumulation of the toxin in the midgut and binding of the toxin to the brush border membranes in previously intoxicated European corn borer larvae.

These results show the surprising benefit of using the CryIB protein in combination with the CryIAb protein or the CryIAc protein, particularly with the CryIAb protein, against Ostrinia, particularly O. nubilalis.

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    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 3                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4074 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bacillus thuringiensis                                           (B) STRAIN: entomocidus HD 110                                                 (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 186..3872                                                        (D) OTHER INFORMATION: /note="PROPERTIES: CryIB is toxic to                    Ostrinea nubilalis (among others)"                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        AAACTGTGGCTAAGATAAATGGCCCTAAATTATGAAGGTATATGTGCTAAAGTCCAAAAA60                 GCGGGAGGTAATTCATCAAAAAATTTTACTATACAATTTTCTTAGGTAATGCTGTGTTAA120                AACTAATCAGTGAAGAAAAGTTAAATAGTTGGTAATATAAGCCCAACATAAAAGGAGGAG180                TTATATTGACTTCAAATAGGAAAAATGAGAATGAAATTATAAATGCT227                             MetThrSerAsnArgLysAsnGluAsnGluIleIleAsnAla                                     1510                                                                           GTATCGAATCATTCCGCACAAATGGATCTATTACCAGATGCTCGTATT275                            ValSerAsnHisSerAlaGlnMetAspLeuLeuProAspAlaArgIle                               15202530                                                                       GAGGATAGCTTGTGTATAGCCGAGGGGAACAATATTGATCCATTTGTT323                            GluAspSerLeuCysIleAlaGluGlyAsnAsnIleAspProPheVal                               354045                                                                         AGCGCATCAACAGTCCAAACGGGTATTAACATAGCTGGTAGAATACTA371                            SerAlaSerThrValGlnThrGlyIleAsnIleAlaGlyArgIleLeu                               505560                                                                         GGCGTATTGGGCGTACCGTTTGCTGGACAACTAGCTAGTTTTTATAGT419                            GlyValLeuGlyValProPheAlaGlyGlnLeuAlaSerPheTyrSer                               657075                                                                         TTTCTTGTTGGTGAATTATGGCCCCGCGGCAGAGATCAGTGGGAAATT467                            PheLeuValGlyGluLeuTrpProArgGlyArgAspGlnTrpGluIle                               808590                                                                         TTCCTAGAACATGTCGAACAACTTATAAATCAACAAATAACAGAAAAT515                            PheLeuGluHisValGluGlnLeuIleAsnGlnGlnIleThrGluAsn                               95100105110                                                                    GCTAGGAATACGGCTCTTGCTCGATTACAAGGTTTAGGAGATTCCTTC563                            AlaArgAsnThrAlaLeuAlaArgLeuGlnGlyLeuGlyAspSerPhe                               115120125                                                                      AGAGCCTATCAACAGTCACTTGAAGATTGGCTAGAAAACCGTGATGAT611                            ArgAlaTyrGlnGlnSerLeuGluAspTrpLeuGluAsnArgAspAsp                               130135140                                                                      GCAAGAACGAGAAGTGTTCTTCATACCCAATATATAGCTTTAGAACTT659                            AlaArgThrArgSerValLeuHisThrGlnTyrIleAlaLeuGluLeu                               145150155                                                                      GATTTTCTTAATGCGATGCCGCTTTTCGCAATTAGAAACCAAGAAGTT707                            AspPheLeuAsnAlaMetProLeuPheAlaIleArgAsnGlnGluVal                               160165170                                                                      CCATTATTGATGGTATATGCTCAAGCTGCAAATTTACACCTATTATTA755                            ProLeuLeuMetValTyrAlaGlnAlaAlaAsnLeuHisLeuLeuLeu                               175180185190                                                                   TTGAGAGATGCCTCTCTTTTTGGTAGTGAATTTGGGCTTACATCGCAG803                            LeuArgAspAlaSerLeuPheGlySerGluPheGlyLeuThrSerGln                               195200205                                                                      GAAATTCAACGCTATTATGAGCGCCAAGTGGAACGAACGAGAGATTAT851                            GluIleGlnArgTyrTyrGluArgGlnValGluArgThrArgAspTyr                               210215220                                                                      TCCGACTATTGCGTAGAATGGTATAATACAGGTCTAAATAGCTTGAGA899                            SerAspTyrCysValGluTrpTyrAsnThrGlyLeuAsnSerLeuArg                               225230235                                                                      GGGACAAATGCCGCAAGTTGGGTACGGTATAATCAATTCCGTAGAGAT947                            GlyThrAsnAlaAlaSerTrpValArgTyrAsnGlnPheArgArgAsp                               240245250                                                                      CTAACGTTAGGAGTATTAGATCTAGTGGCACTATTCCCAAGCTATGAC995                            LeuThrLeuGlyValLeuAspLeuValAlaLeuPheProSerTyrAsp                               255260265270                                                                   ACTCGCACTTATCCAATAAATACGAGTGCTCAGTTAACAAGAGAAGTT1043                           ThrArgThrTyrProIleAsnThrSerAlaGlnLeuThrArgGluVal                               275280285                                                                      TATACAGACGCAATTGGAGCAACAGGGGTAAATATGGCAAGTATGAAT1091                           TyrThrAspAlaIleGlyAlaThrGlyValAsnMetAlaSerMetAsn                               290295300                                                                      TGGTATAATAATAATGCACCTTCGTTCTCTGCCATAGAGGCTGCGGCT1139                           TrpTyrAsnAsnAsnAlaProSerPheSerAlaIleGluAlaAlaAla                               305310315                                                                      ATCCGAAGCCCGCATCTACTTGATTTTCTAGAACAACTTACAATTTTT1187                           IleArgSerProHisLeuLeuAspPheLeuGluGlnLeuThrIlePhe                               320325330                                                                      AGCGCTTCATCACGATGGAGTAATACTAGGCATATGACTTATTGGCGG1235                           SerAlaSerSerArgTrpSerAsnThrArgHisMetThrTyrTrpArg                               335340345350                                                                   GGGCACACGATTCAATCTCGGCCAATAGGAGGCGGATTAAATACCTCA1283                           GlyHisThrIleGlnSerArgProIleGlyGlyGlyLeuAsnThrSer                               355360365                                                                      ACGCATGGGGCTACCAATACTTCTATTAATCCTGTAACATTACGGTTC1331                           ThrHisGlyAlaThrAsnThrSerIleAsnProValThrLeuArgPhe                               370375380                                                                      GCATCTCGAGACGTTTATAGGACTGAATCATATGCAGGAGTGCTTCTA1379                           AlaSerArgAspValTyrArgThrGluSerTyrAlaGlyValLeuLeu                               385390395                                                                      TGGGGAATTTACCTTGAACCTATTCATGGTGTCCCTACTGTTAGGTTT1427                           TrpGlyIleTyrLeuGluProIleHisGlyValProThrValArgPhe                               400405410                                                                      AATTTTACGAACCCTCAGAATATTTCTGATAGAGGTACCGCTAACTAT1475                           AsnPheThrAsnProGlnAsnIleSerAspArgGlyThrAlaAsnTyr                               415420425430                                                                   AGTCAACCTTATGAGTCACCTGGGCTTCAATTAAAAGATTCAGAAACT1523                           SerGlnProTyrGluSerProGlyLeuGlnLeuLysAspSerGluThr                               435440445                                                                      GAATTACCACCAGAAACAACAGAACGACCAAATTATGAATCTTACAGT1571                           GluLeuProProGluThrThrGluArgProAsnTyrGluSerTyrSer                               450455460                                                                      CACAGGTTATCTCATATAGGTATAATTTTACAATCCAGGGTGAATGTA1619                           HisArgLeuSerHisIleGlyIleIleLeuGlnSerArgValAsnVal                               465470475                                                                      CCGGTATATTCTTGGACGCATCGTAGTGCAGATCGTACGAATACGATT1667                           ProValTyrSerTrpThrHisArgSerAlaAspArgThrAsnThrIle                               480485490                                                                      GGACCAAATAGAATCACCCAAATCCCAATGGTAAAAGCATCCGAACTT1715                           GlyProAsnArgIleThrGlnIleProMetValLysAlaSerGluLeu                               495500505510                                                                   CCTCAAGGTACCACTGTTGTTAGAGGACCAGGATTTACTGGTGGGGAT1763                           ProGlnGlyThrThrValValArgGlyProGlyPheThrGlyGlyAsp                               515520525                                                                      ATTCTTCGAAGAACGAATACTGGTGGATTTGGACCGATAAGAGTAACT1811                           IleLeuArgArgThrAsnThrGlyGlyPheGlyProIleArgValThr                               530535540                                                                      GTTAACGGACCATTAACACAAAGATATCGTATAGGATTCCGCTATGCT1859                           ValAsnGlyProLeuThrGlnArgTyrArgIleGlyPheArgTyrAla                               545550555                                                                      TCAACTGTAGATTTTGATTTCTTTGTATCACGTGGAGGTACTACTGTA1907                           SerThrValAspPheAspPhePheValSerArgGlyGlyThrThrVal                               560565570                                                                      AATAATTTTAGATTCCTACGTACAATGAACAGTGGAGACGAACTAAAA1955                           AsnAsnPheArgPheLeuArgThrMetAsnSerGlyAspGluLeuLys                               575580585590                                                                   TACGGAAATTTTGTGAGACGTGCTTTTACTACACCTTTTACTTTTACA2003                           TyrGlyAsnPheValArgArgAlaPheThrThrProPheThrPheThr                               595600605                                                                      CAAATTCAAGATATAATTCGAACGTCTATTCAAGGCCTTAGTGGAAAT2051                           GlnIleGlnAspIleIleArgThrSerIleGlnGlyLeuSerGlyAsn                               610615620                                                                      GGGGAAGTGTATATAGATAAAATTGAAATTATTCCAGTTACTGCAACC2099                           GlyGluValTyrIleAspLysIleGluIleIleProValThrAlaThr                               625630635                                                                      TTCGAAGCAGAATATGATTTAGAAAGAGCGCAAGAGGCGGTGAATGCT2147                           PheGluAlaGluTyrAspLeuGluArgAlaGlnGluAlaValAsnAla                               640645650                                                                      CTGTTTACTAATACGAATCCAAGAAGATTGAAAACAGATGTGACAGAT2195                           LeuPheThrAsnThrAsnProArgArgLeuLysThrAspValThrAsp                               655660665670                                                                   TATCATATTGATCAAGTATCCAATTTAGTGGCGTGTTTATCGGATGAA2243                           TyrHisIleAspGlnValSerAsnLeuValAlaCysLeuSerAspGlu                               675680685                                                                      TTCTGCTTGGATGAAAAGAGAGAATTACTTGAGAAAGTGAAATATGCG2291                           PheCysLeuAspGluLysArgGluLeuLeuGluLysValLysTyrAla                               690695700                                                                      AAACGACTCAGTGATGAAAGAAACTTACTCCAAGATCCAAACTTCACA2339                           LysArgLeuSerAspGluArgAsnLeuLeuGlnAspProAsnPheThr                               705710715                                                                      TCCATCAATAAGCAACCAGACTTCATATCTACTAATGAGCAATCGAAT2387                           SerIleAsnLysGlnProAspPheIleSerThrAsnGluGlnSerAsn                               720725730                                                                      TTCACATCTATCCATGAACAATCTGAACATGGATGGTGGGGAAGTGAG2435                           PheThrSerIleHisGluGlnSerGluHisGlyTrpTrpGlySerGlu                               735740745750                                                                   AACATTACCATCCAGGAAGGAAATGACGTATTTAAAGAGAATTACGTC2483                           AsnIleThrIleGlnGluGlyAsnAspValPheLysGluAsnTyrVal                               755760765                                                                      ACACTACCGGGTACTTTTAATGAGTGTTATCCGACGTATTTATATCAA2531                           ThrLeuProGlyThrPheAsnGluCysTyrProThrTyrLeuTyrGln                               770775780                                                                      AAAATAGGGGAGTCGGAATTAAAAGCTTATACTCGCTACCAATTAAGA2579                           LysIleGlyGluSerGluLeuLysAlaTyrThrArgTyrGlnLeuArg                               785790795                                                                      GGTTATATTGAAGATAGTCAAGATTTAGAGATATATTTGATTCGTTAT2627                           GlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArgTyr                               800805810                                                                      AATGCGAAACATGAAACATTGGATGTTCCAGGTACCGAGTCCCTATGG2675                           AsnAlaLysHisGluThrLeuAspValProGlyThrGluSerLeuTrp                               815820825830                                                                   CCGCTTTCAGTTGAAAGCCCAATCGGAAGGTGCGGAGAACCGAATCGA2723                           ProLeuSerValGluSerProIleGlyArgCysGlyGluProAsnArg                               835840845                                                                      TGCGCACCACATTTTGAATGGAATCCTGATCTAGATTGTTCCTGCAGA2771                           CysAlaProHisPheGluTrpAsnProAspLeuAspCysSerCysArg                               850855860                                                                      GATGGAGAAAAATGTGCGCATCATTCCCATCATTTCTCTTTGGATATT2819                           AspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAspIle                               865870875                                                                      GATGTTGGATGCACAGACTTGCATGAGAATCTAGGCGTGTGGGTGGTA2867                           AspValGlyCysThrAspLeuHisGluAsnLeuGlyValTrpValVal                               880885890                                                                      TTCAAGATTAAGACGCAGGAAGGTCATGCAAGACTAGGGAATCTGGAA2915                           PheLysIleLysThrGlnGluGlyHisAlaArgLeuGlyAsnLeuGlu                               895900905910                                                                   TTTATTGAAGAGAAACCATTATTAGGAGAAGCACTGTCTCGTGTGAAG2963                           PheIleGluGluLysProLeuLeuGlyGluAlaLeuSerArgValLys                               915920925                                                                      AGGGCAGAGAAAAAATGGAGAGACAAACGTGAAAAACTACAATTGGAA3011                           ArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGlnLeuGlu                               930935940                                                                      ACAAAACGAGTATATACAGAGGCAAAAGAAGCTGTGGATGCTTTATTC3059                           ThrLysArgValTyrThrGluAlaLysGluAlaValAspAlaLeuPhe                               945950955                                                                      GTAGATTCTCAATATGATAGATTACAAGCGGATACAAACATCGGCATG3107                           ValAspSerGlnTyrAspArgLeuGlnAlaAspThrAsnIleGlyMet                               960965970                                                                      ATTCATGCGGCAGATAAACTTGTTCATCGAATTCGAGAGGCGTATCTT3155                           IleHisAlaAlaAspLysLeuValHisArgIleArgGluAlaTyrLeu                               975980985990                                                                   TCAGAATTACCTGTTATCCCAGGTGTAAATGCGGAAATTTTTGAAGAA3203                           SerGluLeuProValIleProGlyValAsnAlaGluIlePheGluGlu                               99510001005                                                                    TTAGAAGGTCACATTATCACTGCAATCTCCTTATACGATGCGAGAAAT3251                           LeuGluGlyHisIleIleThrAlaIleSerLeuTyrAspAlaArgAsn                               101010151020                                                                   GTCGTTAAAAATGGTGATTTTAATAATGGATTAACATGTTGGAATGTA3299                           ValValLysAsnGlyAspPheAsnAsnGlyLeuThrCysTrpAsnVal                               102510301035                                                                   AAAGGGCATGTAGATGTACAACAGAGCCATCATCGTTCTGACCTTGTT3347                           LysGlyHisValAspValGlnGlnSerHisHisArgSerAspLeuVal                               104010451050                                                                   ATCCCAGAATGGGAAGCAGAAGTGTCACAAGCAGTTCGCGTCTGTCCG3395                           IleProGluTrpGluAlaGluValSerGlnAlaValArgValCysPro                               1055106010651070                                                               GGGTGTGGCTATATCCTTCGTGTCACAGCGTACAAAGAGGGATATGGA3443                           GlyCysGlyTyrIleLeuArgValThrAlaTyrLysGluGlyTyrGly                               107510801085                                                                   GAGGGCTGCGTAACGATCCATGAAATCGAGAACAATACAGACGAACTA3491                           GluGlyCysValThrIleHisGluIleGluAsnAsnThrAspGluLeu                               109010951100                                                                   AAATTTAAAAACCGTGAAGAAGAGGAAGTGTATCCAACGGATACAGGA3539                           LysPheLysAsnArgGluGluGluGluValTyrProThrAspThrGly                               110511101115                                                                   ACGTGTAATGATTATACTGCACACCAAGGTACAGCTGGATGCGCAGAT3587                           ThrCysAsnAspTyrThrAlaHisGlnGlyThrAlaGlyCysAlaAsp                               112011251130                                                                   GCATGTAATTCCCGTAATGCTGGATATGAGGATGCATATGAAGTTGAT3635                           AlaCysAsnSerArgAsnAlaGlyTyrGluAspAlaTyrGluValAsp                               1135114011451150                                                               ACTACAGCATCTGTTAATTACAAACCGACTTATGAAGAAGAAACGTAT3683                           ThrThrAlaSerValAsnTyrLysProThrTyrGluGluGluThrTyr                               115511601165                                                                   ACAGATGTAAGAAGAGATAATCATTGTGAATATGACAGAGGGTATGTC3731                           ThrAspValArgArgAspAsnHisCysGluTyrAspArgGlyTyrVal                               117011751180                                                                   AATTATCCACCAGTACCAGCTGGTTATGTGACAAAAGAATTAGAATAC3779                           AsnTyrProProValProAlaGlyTyrValThrLysGluLeuGluTyr                               118511901195                                                                   TTCCCAGAAACAGATACAGTATGGATTGAGATTGGAGAAACGGAAGGA3827                           PheProGluThrAspThrValTrpIleGluIleGlyGluThrGluGly                               120012051210                                                                   AAGTTTATTGTAGATAGCGTGGAATTACTCCTCATGGAAGAATAGGATCATC3879                       LysPheIleValAspSerValGluLeuLeuLeuMetGluGlu                                     121512201225                                                                   CAAGTATAGCAGTTTAATAAATATTAATTAAAATAGTAGTCTAACTTCCGTTCCAATTAA3939               ATAAGTAAATTACAGTTGTAAAAAGAAAACGGACATCACTCTTCAGAGAGCGATGTCCGT3999               TTTTTATATGGTTTGTGCTAATGATAAGTGTGCACGAAATTTTATTGTCAAAATAGTATT4059               TACTTGAGAAAAAGA4074                                                            (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 4343 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bacillus thuringiensis                                           (B) STRAIN: berliner 1715                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 141..3608                                                        (D) OTHER INFORMATION: /note="coding sequence for CryIAb                       insecticidal crystal protein                                                   PROPERTIES: CryIAb is toxic to Ostrinia nubilalis                              (amongothers)"                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        CAAAAATTGATATTTAGTAAAATTAGTTGCACTTTGTGCATTTTTTCATAAGATGAGTCA60                 TATGTTTTAAATTGTAGTAATGAAAAACAGTATTATATCATAATGAATTGGTATCTTAAT120                AAAAGAGATGGAGGTAACTTATGGATAACAATCCGAACATCAATGAATGC170                          MetAspAsnAsnProAsnIleAsnGluCys                                                 1510                                                                           ATTCCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATTAGGTGGA218                            IleProTyrAsnCysLeuSerAsnProGluValGluValLeuGlyGly                               152025                                                                         GAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTGTCGCTA266                            GluArgIleGluThrGlyTyrThrProIleAspIleSerLeuSerLeu                               303540                                                                         ACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTA314                            ThrGlnPheLeuLeuSerGluPheValProGlyAlaGlyPheValLeu                               455055                                                                         GGACTAGTTGATATAATATGGGGAATTTTTGGTCCCTCTCAATGGGAC362                            GlyLeuValAspIleIleTrpGlyIlePheGlyProSerGlnTrpAsp                               606570                                                                         GCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAATAGAAGAA410                            AlaPheLeuValGlnIleGluGlnLeuIleAsnGlnArgIleGluGlu                               75808590                                                                       TTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTT458                            PheAlaArgAsnGlnAlaIleSerArgLeuGluGlyLeuSerAsnLeu                               95100105                                                                       TATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACT506                            TyrGlnIleTyrAlaGluSerPheArgGluTrpGluAlaAspProThr                               110115120                                                                      AATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAAC554                            AsnProAlaLeuArgGluGluMetArgIleGlnPheAsnAspMetAsn                               125130135                                                                      AGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAA602                            SerAlaLeuThrThrAlaIleProLeuPheAlaValGlnAsnTyrGln                               140145150                                                                      GTTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTATCA650                            ValProLeuLeuSerValTyrValGlnAlaAlaAsnLeuHisLeuSer                               155160165170                                                                   GTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCC698                            ValLeuArgAspValSerValPheGlyGlnArgTrpGlyPheAspAla                               175180185                                                                      GCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAAC746                            AlaThrIleAsnSerArgTyrAsnAspLeuThrArgLeuIleGlyAsn                               190195200                                                                      TATACAGATCATGCTGTACGCTGGTACAATACGGGATTAGAGCGTGTA794                            TyrThrAspHisAlaValArgTrpTyrAsnThrGlyLeuGluArgVal                               205210215                                                                      TGGGGACCGGATTCTAGAGATTGGATAAGATATAATCAATTTAGAAGA842                            TrpGlyProAspSerArgAspTrpIleArgTyrAsnGlnPheArgArg                               220225230                                                                      GAATTAACACTAACTGTATTAGATATCGTTTCTCTATTTCCGAACTAT890                            GluLeuThrLeuThrValLeuAspIleValSerLeuPheProAsnTyr                               235240245250                                                                   GATAGTAGAACGTATCCAATTCGAACAGTTTCCCAATTAACAAGAGAA938                            AspSerArgThrTyrProIleArgThrValSerGlnLeuThrArgGlu                               255260265                                                                      ATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGAGGC986                            IleTyrThrAsnProValLeuGluAsnPheAspGlySerPheArgGly                               270275280                                                                      TCGGCTCAGGGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGAT1034                           SerAlaGlnGlyIleGluGlySerIleArgSerProHisLeuMetAsp                               285290295                                                                      ATACTTAACAGTATAACCATCTATACGGATGCTCATAGAGGAGAATAT1082                           IleLeuAsnSerIleThrIleTyrThrAspAlaHisArgGlyGluTyr                               300305310                                                                      TATTGGTCAGGGCATCAAATAATGGCTTCTCCTGTAGGGTTTTCGGGG1130                           TyrTrpSerGlyHisGlnIleMetAlaSerProValGlyPheSerGly                               315320325330                                                                   CCAGAATTCACTTTTCCGCTATATGGAACTATGGGAAATGCAGCTCCA1178                           ProGluPheThrPheProLeuTyrGlyThrMetGlyAsnAlaAlaPro                               335340345                                                                      CAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTA1226                           GlnGlnArgIleValAlaGlnLeuGlyGlnGlyValTyrArgThrLeu                               350355360                                                                      TCGTCCACTTTATATAGAAGACCTTTTAATATAGGGATAAATAATCAA1274                           SerSerThrLeuTyrArgArgProPheAsnIleGlyIleAsnAsnGln                               365370375                                                                      CAACTATCTGTTCTTGACGGGACAGAATTTGCTTATGGAACCTCCTCA1322                           GlnLeuSerValLeuAspGlyThrGluPheAlaTyrGlyThrSerSer                               380385390                                                                      AATTTGCCATCCGCTGTATACAGAAAAAGCGGAACGGTAGATTCGCTG1370                           AsnLeuProSerAlaValTyrArgLysSerGlyThrValAspSerLeu                               395400405410                                                                   GATGAAATACCGCCACAGAATAACAACGTGCCACCTAGGCAAGGATTT1418                           AspGluIleProProGlnAsnAsnAsnValProProArgGlnGlyPhe                               415420425                                                                      AGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTTAGTAAT1466                           SerHisArgLeuSerHisValSerMetPheArgSerGlyPheSerAsn                               430435440                                                                      AGTAGTGTAAGTATAATAAGAGCTCCTATGTTCTCTTGGATACATCGT1514                           SerSerValSerIleIleArgAlaProMetPheSerTrpIleHisArg                               445450455                                                                      AGTGCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATA1562                           SerAlaGluPheAsnAsnIleIleProSerSerGlnIleThrGlnIle                               460465470                                                                      CCTTTAACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAA1610                           ProLeuThrLysSerThrAsnLeuGlySerGlyThrSerValValLys                               475480485490                                                                   GGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGC1658                           GlyProGlyPheThrGlyGlyAspIleLeuArgArgThrSerProGly                               495500505                                                                      CAGATTTCAACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGA1706                           GlnIleSerThrLeuArgValAsnIleThrAlaProLeuSerGlnArg                               510515520                                                                      TATCGGGTAAGAATTCGCTACGCTTCTACCACAAATTTACAATTCCAT1754                           TyrArgValArgIleArgTyrAlaSerThrThrAsnLeuGlnPheHis                               525530535                                                                      ACATCAATTGACGGAAGACCTATTAATCAGGGGAATTTTTCAGCAACT1802                           ThrSerIleAspGlyArgProIleAsnGlnGlyAsnPheSerAlaThr                               540545550                                                                      ATGAGTAGTGGGAGTAATTTACAGTCCGGAAGCTTTAGGACTGTAGGT1850                           MetSerSerGlySerAsnLeuGlnSerGlySerPheArgThrValGly                               555560565570                                                                   TTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTA1898                           PheThrThrProPheAsnPheSerAsnGlySerSerValPheThrLeu                               575580585                                                                      AGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTATATAGATCGAATT1946                           SerAlaHisValPheAsnSerGlyAsnGluValTyrIleAspArgIle                               590595600                                                                      GAATTTGTTCCGGCAGAAGTAACCTTTGAGGCAGAATATGATTTAGAA1994                           GluPheValProAlaGluValThrPheGluAlaGluTyrAspLeuGlu                               605610615                                                                      AGAGCACAAAAGGCGGTGAATGAGCTGTTTACTTCTTCCAATCAAATC2042                           ArgAlaGlnLysAlaValAsnGluLeuPheThrSerSerAsnGlnIle                               620625630                                                                      GGGTTAAAAACAGATGTGACGGATTATCATATTGATCAAGTATCCAAT2090                           GlyLeuLysThrAspValThrAspTyrHisIleAspGlnValSerAsn                               635640645650                                                                   TTAGTTGAGTGTTTATCTGATGAATTTTGTCTGGATGAAAAAAAAGAA2138                           LeuValGluCysLeuSerAspGluPheCysLeuAspGluLysLysGlu                               655660665                                                                      TTGTCCGAGAAAGTCAAACATGCGAAGCGACTTAGTGATGAGCGGAAT2186                           LeuSerGluLysValLysHisAlaLysArgLeuSerAspGluArgAsn                               670675680                                                                      TTACTTCAAGATCCAAACTTTAGAGGGATCAATAGACAACTAGACCGT2234                           LeuLeuGlnAspProAsnPheArgGlyIleAsnArgGlnLeuAspArg                               685690695                                                                      GGCTGGAGAGGAAGTACGGATATTACCATCCAAGGAGGCGATGACGTA2282                           GlyTrpArgGlySerThrAspIleThrIleGlnGlyGlyAspAspVal                               700705710                                                                      TTCAAAGAGAATTACGTTACGCTATTGGGTACCTTTGATGAGTGCTAC2330                           PheLysGluAsnTyrValThrLeuLeuGlyThrPheAspGluCysTyr                               715720725730                                                                   TTAACGTATTTATATCAAAAAATAGATGAGTCGAAATTAAAAGCCTAT2378                           LeuThrTyrLeuTyrGlnLysIleAspGluSerLysLeuLysAlaTyr                               735740745                                                                      ACCCGTTACCAATTAAGAGGGTATATCGAAGATAGTCAAGACTTAGAA2426                           ThrArgTyrGlnLeuArgGlyTyrIleGluAspSerGlnAspLeuGlu                               750755760                                                                      ATCTATTTAATTCGCTACAATGCCAAACACGAAACAGTAAATGTGCCA2474                           IleTyrLeuIleArgTyrAsnAlaLysHisGluThrValAsnValPro                               765770775                                                                      GGTACGGGTTCCTTATGGCGCCTTTCAGCCCCAAGTCCAATCGGAAAA2522                           GlyThrGlySerLeuTrpArgLeuSerAlaProSerProIleGlyLys                               780785790                                                                      TGTGCCCATCATTCCCATCATTTCTCCTTGGACATTGATGTTGGATGT2570                           CysAlaHisHisSerHisHisPheSerLeuAspIleAspValGlyCys                               795800805810                                                                   ACAGACTTAAATGAGGACTTAGGTGTATGGGTGATATTCAAGATTAAG2618                           ThrAspLeuAsnGluAspLeuGlyValTrpValIlePheLysIleLys                               815820825                                                                      ACGCAAGATGGCCATGCAAGACTAGGAAATCTAGAATTTCTCGAAGAG2666                           ThrGlnAspGlyHisAlaArgLeuGlyAsnLeuGluPheLeuGluGlu                               830835840                                                                      AAACCATTAGTAGGAGAAGCACTAGCTCGTGTGAAAAGAGCGGAGAAA2714                           LysProLeuValGlyGluAlaLeuAlaArgValLysArgAlaGluLys                               845850855                                                                      AAATGGAGAGACAAACGTGAAAAATTGGAATGGGAAACAAATATTGTT2762                           LysTrpArgAspLysArgGluLysLeuGluTrpGluThrAsnIleVal                               860865870                                                                      TATAAAGAGGCAAAAGAATCTGTAGATGCTTTATTTGTAAACTCTCAA2810                           TyrLysGluAlaLysGluSerValAspAlaLeuPheValAsnSerGln                               875880885890                                                                   TATGATAGATTACAAGCGGATACCAACATCGCGATGATTCATGCGGCA2858                           TyrAspArgLeuGlnAlaAspThrAsnIleAlaMetIleHisAlaAla                               895900905                                                                      GATAAACGCGTTCATAGCATTCGAGAAGCTTATCTGCCTGAGCTGTCT2906                           AspLysArgValHisSerIleArgGluAlaTyrLeuProGluLeuSer                               910915920                                                                      GTGATTCCGGGTGTCAATGCGGCTATTTTTGAAGAATTAGAAGGGCGT2954                           ValIleProGlyValAsnAlaAlaIlePheGluGluLeuGluGlyArg                               925930935                                                                      ATTTTCACTGCATTCTCCCTATATGATGCGAGAAATGTCATTAAAAAT3002                           IlePheThrAlaPheSerLeuTyrAspAlaArgAsnValIleLysAsn                               940945950                                                                      GGTGATTTTAATAATGGCTTATCCTGCTGGAACGTGAAAGGGCATGTA3050                           GlyAspPheAsnAsnGlyLeuSerCysTrpAsnValLysGlyHisVal                               955960965970                                                                   GATGTAGAAGAACAAAACAACCACCGTTCGGTCCTTGTTGTTCCGGAA3098                           AspValGluGluGlnAsnAsnHisArgSerValLeuValValProGlu                               975980985                                                                      TGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGGTCGTGGC3146                           TrpGluAlaGluValSerGlnGluValArgValCysProGlyArgGly                               9909951000                                                                     TATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAGGTTGC3194                           TyrIleLeuArgValThrAlaTyrLysGluGlyTyrGlyGluGlyCys                               100510101015                                                                   GTAACCATTCATGAGATCGAGAACAATACAGACGAACTGAAGTTTAGC3242                           ValThrIleHisGluIleGluAsnAsnThrAspGluLeuLysPheSer                               102010251030                                                                   AACTGTGTAGAAGAGGAAGTATATCCAAACAACACGGTAACGTGTAAT3290                           AsnCysValGluGluGluValTyrProAsnAsnThrValThrCysAsn                               1035104010451050                                                               GATTATACTGCGACTCAAGAAGAATATGAGGGTACGTACACTTCTCGT3338                           AspTyrThrAlaThrGlnGluGluTyrGluGlyThrTyrThrSerArg                               105510601065                                                                   AATCGAGGATATGACGGAGCCTATGAAAGCAATTCTTCTGTACCAGCT3386                           AsnArgGlyTyrAspGlyAlaTyrGluSerAsnSerSerValProAla                               107010751080                                                                   GATTATGCATCAGCCTATGAAGAAAAAGCATATACAGATGGACGAAGA3434                           AspTyrAlaSerAlaTyrGluGluLysAlaTyrThrAspGlyArgArg                               108510901095                                                                   GACAATCCTTGTGAATCTAACAGAGGATATGGGGATTACACACCACTA3482                           AspAsnProCysGluSerAsnArgGlyTyrGlyAspTyrThrProLeu                               110011051110                                                                   CCAGCTGGCTATGTGACAAAAGAATTAGAGTACTTCCCAGAAACCGAT3530                           ProAlaGlyTyrValThrLysGluLeuGluTyrPheProGluThrAsp                               1115112011251130                                                               AAGGTATGGATTGAGATCGGAGAAACGGAAGGAACATTCATCGTGGAC3578                           LysValTrpIleGluIleGlyGluThrGluGlyThrPheIleValAsp                               113511401145                                                                   AGCGTGGAATTACTTCTTATGGAGGAATAATATATGCTTTAAAATGT3625                            SerValGluLeuLeuLeuMetGluGlu                                                    11501155                                                                       AAGGTGTGCAAATAAAGAATGATTACTGACTTGTATTGACAGATAAATAAGGAAATTTTT3685               ATATGAATAAAAAACGGGCATCACTCTTAAAAGAATGATGTCCGTTTTTTGTATGATTTA3745               ACGAGTGATATTTAAATGTTTTTTTGCGAAGGCTTTACTTAACGGGGTACCGCCACATGC3805               CCATCAACTTAAGAATTTGCACTACCCCCAAGTGTCAAAAAACGTTATTCTTTCTAAAAA3865               GCTAGCTAGAAAGGATGACATTTTTTATGAATCTTTCAATTCAAGATGAATTACAACTAT3925               TTTCTGAAGAGCTGTATCGTCATTTAACCCCTTCTCTTTTGGAAGAACTCGCTAAAGAAT3985               TAGGTTTTGTAAAAAGAAAACGAAAGTTTTCAGGAAATGAATTAGCTACCATATGTATCT4045               GGGTCAGTCAACGTACAGCGAGTGATTCTCTCGTTCGACTATGCAGTCAATTACACGCCG4105               CCACAGGACCTCTTATGAGTCCAGAAGGACTCAATAAACGCTTTGATAAAAAAGCGGTTG4165               AATTTTTGAAATATATTTTTTCTGCATTATGGAAAAGTAAACTTTGTAAAACATCAGCCA4225               TTTCAAGTGCAGCACTCACGTATTTTCAACGAATCCGTATTTTAGATGCGACGATTTTCC4285               AAGTACCGAAACATTTAGCACATGTATATCCTGGGTCAGGTGGTTGTGCACAAACTGC4343                 (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3537 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bacillus thuringiensis                                           (B) STRAIN: kurstaki HD-73                                                     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..3537                                                          (D) OTHER INFORMATION: /note="FEATURES: sequence encodes                       CryIAc insecticidal crystal protein                                            PROPERTIES: CryIAc is toxic to Ostrinia nubilalis                              (amongothers)                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATGGATAACAATCCGAACATCAATGAATGCATTCCTTATAATTGTTTA48                             MetAspAsnAsnProAsnIleAsnGluCysIleProTyrAsnCysLeu                               151015                                                                         AGTAACCCTGAAGTAGAAGTATTAGGTGGAGAAAGAATAGAAACTGGT96                             SerAsnProGluValGluValLeuGlyGlyGluArgIleGluThrGly                               202530                                                                         TACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTTCTTTTGAGT144                            TyrThrProIleAspIleSerLeuSerLeuThrGlnPheLeuLeuSer                               354045                                                                         GAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATA192                            GluPheValProGlyAlaGlyPheValLeuGlyLeuValAspIleIle                               505560                                                                         TGGGGAATTTTTGGTCCCTCTCAATGGGACGCATTTCTTGTACAAATT240                            TrpGlyIlePheGlyProSerGlnTrpAspAlaPheLeuValGlnIle                               65707580                                                                       GAACAGTTAATTAACCAAAGAATAGAAGAATTCGCTAGGAACCAAGCC288                            GluGlnLeuIleAsnGlnArgIleGluGluPheAlaArgAsnGlnAla                               859095                                                                         ATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTTACGCAGAA336                            IleSerArgLeuGluGlyLeuSerAsnLeuTyrGlnIleTyrAlaGlu                               100105110                                                                      TCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAA384                            SerPheArgGluTrpGluAlaAspProThrAsnProAlaLeuArgGlu                               115120125                                                                      GAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCT432                            GluMetArgIleGlnPheAsnAspMetAsnSerAlaLeuThrThrAla                               130135140                                                                      ATTCCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTA480                            IleProLeuPheAlaValGlnAsnTyrGlnValProLeuLeuSerVal                               145150155160                                                                   TATGTTCAAGCTGCAAATTTACATTTATCAGTTTTGAGAGATGTTTCA528                            TyrValGlnAlaAlaAsnLeuHisLeuSerValLeuArgAspValSer                               165170175                                                                      GTGTTTGGACAAAGGTGGGGATTTGATGCCGCGACTATCAATAGTCGT576                            ValPheGlyGlnArgTrpGlyPheAspAlaAlaThrIleAsnSerArg                               180185190                                                                      TATAATGATTTAACTAGGCTTATTGGCAACTATACAGATTATGCTGTA624                            TyrAsnAspLeuThrArgLeuIleGlyAsnTyrThrAspTyrAlaVal                               195200205                                                                      CGCTGGTACAATACGGGATTAGAACGTGTATGGGGACCGGATTCTAGA672                            ArgTrpTyrAsnThrGlyLeuGluArgValTrpGlyProAspSerArg                               210215220                                                                      GATTGGGTAAGGTATAATCAATTTAGAAGAGAATTAACACTAACTGTA720                            AspTrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrVal                               225230235240                                                                   TTAGATATCGTTGCTCTGTTCCCGAATTATGATAGTAGAAGATATCCA768                            LeuAspIleValAlaLeuPheProAsnTyrAspSerArgArgTyrPro                               245250255                                                                      ATTCGAACAGTTTCCCAATTAACAAGAGAAATTTATACAAACCCAGTA816                            IleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnProVal                               260265270                                                                      TTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGAA864                            LeuGluAsnPheAspGlySerPheArgGlySerAlaGlnGlyIleGlu                               275280285                                                                      AGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACC912                            ArgSerIleArgSerProHisLeuMetAspIleLeuAsnSerIleThr                               290295300                                                                      ATCTATACGGATGCTCATAGGGGTTATTATTATTGGTCAGGGCATCAA960                            IleTyrThrAspAlaHisArgGlyTyrTyrTyrTrpSerGlyHisGln                               305310315320                                                                   ATAATGGCTTCTCCTGTAGGGTTTTCGGGGCCAGAATTCACTTTTCCG1008                           IleMetAlaSerProValGlyPheSerGlyProGluPheThrPhePro                               325330335                                                                      CTATATGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCT1056                           LeuTyrGlyThrMetGlyAsnAlaAlaProGlnGlnArgIleValAla                               340345350                                                                      CAACTAGGTCAGGGCGTGTATAGAACATTATCGTCCACTTTATATAGA1104                           GlnLeuGlyGlnGlyValTyrArgThrLeuSerSerThrLeuTyrArg                               355360365                                                                      AGACCTTTTAATATAGGGATAAATAATCAACAACTATCTGTTCTTGAC1152                           ArgProPheAsnIleGlyIleAsnAsnGlnGlnLeuSerValLeuAsp                               370375380                                                                      GGGACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTA1200                           GlyThrGluPheAlaTyrGlyThrSerSerAsnLeuProSerAlaVal                               385390395400                                                                   TACAGAAAAAGCGGAACGGTAGATTCGCTGGATGAAATACCGCCACAG1248                           TyrArgLysSerGlyThrValAspSerLeuAspGluIleProProGln                               405410415                                                                      AATAACAACGTGCCACCTAGGCAAGGATTTAGTCATCGATTAAGCCAT1296                           AsnAsnAsnValProProArgGlnGlyPheSerHisArgLeuSerHis                               420425430                                                                      GTTTCAATGTTTCGTTCAGGCTTTAGTAATAGTAGTGTAAGTATAATA1344                           ValSerMetPheArgSerGlyPheSerAsnSerSerValSerIleIle                               435440445                                                                      AGAGCTCCTATGTTCTCTTGGATACATCGTAGTGCTGAATTTAATAAT1392                           ArgAlaProMetPheSerTrpIleHisArgSerAlaGluPheAsnAsn                               450455460                                                                      ATAATTGCATCGGATAGTATTACTCAAATCCCTGCAGTGAAGGGAAAC1440                           IleIleAlaSerAspSerIleThrGlnIleProAlaValLysGlyAsn                               465470475480                                                                   TTTCTTTTTAATGGTTCTGTAATTTCAGGACCAGGATTTACTGGTGGG1488                           PheLeuPheAsnGlySerValIleSerGlyProGlyPheThrGlyGly                               485490495                                                                      GACTTAGTTAGATTAAATAGTAGTGGAAATAACATTCAGAATAGAGGG1536                           AspLeuValArgLeuAsnSerSerGlyAsnAsnIleGlnAsnArgGly                               500505510                                                                      TATATTGAAGTTCCAATTCACTTCCCATCGACATCTACCAGATATCGA1584                           TyrIleGluValProIleHisPheProSerThrSerThrArgTyrArg                               515520525                                                                      GTTCGTGTACGGTATGCTTCTGTAACCCCGATTCACCTCAACGTTAAT1632                           ValArgValArgTyrAlaSerValThrProIleHisLeuAsnValAsn                               530535540                                                                      TGGGGTAATTCATCCATTTTTTCCAATACAGTACCAGCTACAGCTACG1680                           TrpGlyAsnSerSerIlePheSerAsnThrValProAlaThrAlaThr                               545550555560                                                                   TCATTAGATAATCTACAATCAAGTGATTTTGGTTATTTTGAAAGTGCC1728                           SerLeuAspAsnLeuGlnSerSerAspPheGlyTyrPheGluSerAla                               565570575                                                                      AATGCTTTTACATCTTCATTAGGTAATATAGTAGGTGTTAGAAATTTT1776                           AsnAlaPheThrSerSerLeuGlyAsnIleValGlyValArgAsnPhe                               580585590                                                                      AGTGGGACTGCAGGAGTGATAATAGACAGATTTGAATTTATTCCAGTT1824                           SerGlyThrAlaGlyValIleIleAspArgPheGluPheIleProVal                               595600605                                                                      ACTGCAACACTCGAGGCTGAATATAATCTGGAAAGAGCGCAGAAGGCG1872                           ThrAlaThrLeuGluAlaGluTyrAsnLeuGluArgAlaGlnLysAla                               610615620                                                                      GTGAATGCGCTGTTTACGTCTACAAACCAACTAGGGCTAAAAACAAAT1920                           ValAsnAlaLeuPheThrSerThrAsnGlnLeuGlyLeuLysThrAsn                               625630635640                                                                   GTAACGGATTATCATATTGATCAAGTGTCCAATTTAGTTACGTATTTA1968                           ValThrAspTyrHisIleAspGlnValSerAsnLeuValThrTyrLeu                               645650655                                                                      TCGGATGAATTTTGTCTGGATGAAAAGCGAGAATTGTCCGAGAAAGTC2016                           SerAspGluPheCysLeuAspGluLysArgGluLeuSerGluLysVal                               660665670                                                                      AAACATGCGAAGCGACTCAGTGATGAACGCAATTTACTCCAAGATTCA2064                           LysHisAlaLysArgLeuSerAspGluArgAsnLeuLeuGlnAspSer                               675680685                                                                      AATTTCAAAGACATTAATAGGCAACCAGAACGTGGGTGGGGCGGAAGT2112                           AsnPheLysAspIleAsnArgGlnProGluArgGlyTrpGlyGlySer                               690695700                                                                      ACAGGGATTACCATCCAAGGAGGGGATGACGTATTTAAAGAAAATTAC2160                           ThrGlyIleThrIleGlnGlyGlyAspAspValPheLysGluAsnTyr                               705710715720                                                                   GTCACACTATCAGGTACCTTTGATGAGTGCTATCCAACATATTTGTAT2208                           ValThrLeuSerGlyThrPheAspGluCysTyrProThrTyrLeuTyr                               725730735                                                                      CAAAAAATCGATGAATCAAAATTAAAAGCCTTTACCCGTTATCAATTA2256                           GlnLysIleAspGluSerLysLeuLysAlaPheThrArgTyrGlnLeu                               740745750                                                                      AGAGGGTATATCGAAGATAGTCAAGACTTAGAAATCTATTTAATTCGC2304                           ArgGlyTyrIleGluAspSerGlnAspLeuGluIleTyrLeuIleArg                               755760765                                                                      TACAATGCAAAACATGAAACAGTAAATGTGCCAGGTACGGGTTCCTTA2352                           TyrAsnAlaLysHisGluThrValAsnValProGlyThrGlySerLeu                               770775780                                                                      TGGCCGCTTTCAGCCCAAAGTCCAATCGGAAAGTGTGGAGAGCCGAAT2400                           TrpProLeuSerAlaGlnSerProIleGlyLysCysGlyGluProAsn                               785790795800                                                                   CGATGCGCGCCACACCTTGAATGGAATCCTGACTTAGATTGTTCGTGT2448                           ArgCysAlaProHisLeuGluTrpAsnProAspLeuAspCysSerCys                               805810815                                                                      AGGGATGGAGAAAAGTGTGCCCATCATTCGCATCATTTCTCCTTAGAC2496                           ArgAspGlyGluLysCysAlaHisHisSerHisHisPheSerLeuAsp                               820825830                                                                      ATTGATGTAGGATGTACAGACTTAAATGAGGACCTAGGTGTATGGGTG2544                           IleAspValGlyCysThrAspLeuAsnGluAspLeuGlyValTrpVal                               835840845                                                                      ATCTTTAAGATTAAGACGCAAGATGGGCACGCAAGACTAGGGAATCTA2592                           IlePheLysIleLysThrGlnAspGlyHisAlaArgLeuGlyAsnLeu                               850855860                                                                      GAGTTTCTCGAAGAGAAACCATTAGTAGGAGAAGCGCTAGCTCGTGTG2640                           GluPheLeuGluGluLysProLeuValGlyGluAlaLeuAlaArgVal                               865870875880                                                                   AAAAGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAATTGGAATGG2688                           LysArgAlaGluLysLysTrpArgAspLysArgGluLysLeuGluTrp                               885890895                                                                      GAAACAAATATCGTTTATAAAGAGGCAAAAGAATCTGTAGATGCTTTA2736                           GluThrAsnIleValTyrLysGluAlaLysGluSerValAspAlaLeu                               900905910                                                                      TTTGTAAACTCTCAATATGATCAATTACAAGCGGATACGAATATTGCC2784                           PheValAsnSerGlnTyrAspGlnLeuGlnAlaAspThrAsnIleAla                               915920925                                                                      ATGATTCATGCGGCAGATAAACGTGTTCATAGCATTCGAGAAGCTTAT2832                           MetIleHisAlaAlaAspLysArgValHisSerIleArgGluAlaTyr                               930935940                                                                      CTGCCTGAGCTGTCTGTGATTCCGGGTGTCAATGCGGCTATTTTTGAA2880                           LeuProGluLeuSerValIleProGlyValAsnAlaAlaIlePheGlu                               945950955960                                                                   GAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGA2928                           GluLeuGluGlyArgIlePheThrAlaPheSerLeuTyrAspAlaArg                               965970975                                                                      AATGTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAAC2976                           AsnValIleLysAsnGlyAspPheAsnAsnGlyLeuSerCysTrpAsn                               980985990                                                                      GTGAAAGGGCATGTAGATGTAGAAGAACAAAACAACCAACGTTCGGTC3024                           ValLysGlyHisValAspValGluGluGlnAsnAsnGlnArgSerVal                               99510001005                                                                    CTTGTTGTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTC3072                           LeuValValProGluTrpGluAlaGluValSerGlnGluValArgVal                               101010151020                                                                   TGTCCGGGTCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGA3120                           CysProGlyArgGlyTyrIleLeuArgValThrAlaTyrLysGluGly                               1025103010351040                                                               TATGGAGAAGGTTGCGTAACCATTCATGAGATCGAGAACAATACAGAC3168                           TyrGlyGluGlyCysValThrIleHisGluIleGluAsnAsnThrAsp                               104510501055                                                                   GAACTGAAGTTTAGCAACTGCGTAGAAGAGGAAATCTATCCAAATAAC3216                           GluLeuLysPheSerAsnCysValGluGluGluIleTyrProAsnAsn                               106010651070                                                                   ACGGTAACGTGTAATGATTATACTGTAAATCAAGAAGAATACGGAGGT3264                           ThrValThrCysAsnAspTyrThrValAsnGlnGluGluTyrGlyGly                               107510801085                                                                   GCGTACACTTCTCGTAATCGAGGATATAACGAAGCTCCTTCCGTACCA3312                           AlaTyrThrSerArgAsnArgGlyTyrAsnGluAlaProSerValPro                               109010951100                                                                   GCTGATTATGCGTCAGTCTATGAAGAAAAATCGTATACAGATGGACGA3360                           AlaAspTyrAlaSerValTyrGluGluLysSerTyrThrAspGlyArg                               1105111011151120                                                               AGAGAGAATCCTTGTGAATTTAACAGAGGGTATAGGGATTACACGCCA3408                           ArgGluAsnProCysGluPheAsnArgGlyTyrArgAspTyrThrPro                               112511301135                                                                   CTACCAGTTGGTTATGTGACAAAAGAATTAGAATACTTCCCAGAAACC3456                           LeuProValGlyTyrValThrLysGluLeuGluTyrPheProGluThr                               114011451150                                                                   GATAAGGTATGGATTGAGATTGGAGAAACGGAAGGAACATTTATCGTG3504                           AspLysValTrpIleGluIleGlyGluThrGluGlyThrPheIleVal                               115511601165                                                                   GACAGCGTGGAATTACTCCTTATGGAGGAATAG3537                                          AspSerValGluLeuLeuLeuMetGluGlu                                                 11701175                                                                       __________________________________________________________________________ 

We claim:
 1. A method to combat or control Ostrinia nubilalis, comprising contacting said Ostrinia nubilalis witha CryIB protein, wherein said CryIB protein comprises the amino acid sequence of SEQ ID NO. 1; an insecticidal fragment of a CryIB protein, wherein said insecticidal fragment of a CryIB protein comprises the amino acid sequence of SEQ ID NO. 1 from amino acid position 1 to amino acid position 636; or variants thereof wherein His at position 150 is replaced by Tyr.
 2. The method according to claim 1, wherein said Ostrinia nubilalis is further contacted with a protein selected from the group consisting of:an insecticidal fragment of a CryIAb protein comprising the amino acid sequence of SEQ ID NO. 2 from amino acid position 29 to amino acid position 607 or a variant thereof having at least one mutation, wherein said at least one mutation comprises Asp at position 542 being replaced by His; Thr at position 568 being replaced by His; Val at position 569 being replaced by Leu; Gly at position 282 being replaced by Ala; Ser at position 283 being replaced by Leu; Ala at position 450 being replaced by Pro; Phe at position 537 being replaced by Leu; and Pro at position 545 being replaced by Ile; and an insecticidal fragment of a CryIAc protein comprising the amino acid sequence of SEQ ID No. 3 from amino acid position 1 to amino acid position 609 or a variant thereof having at least one mutation, wherein said at least one mutation comprises Phe at position 148 being replaced by Leu; Leu at position 366 being replaced by Phe; Phe at position 440 being deleted; and Asn at position 442 being replaced by Ser.
 3. The method according to claim 2, wherein said variant of the CryIAb insecticidal fragment is selected from the group consisting of: a variant in which Asp, Thr and Val, respectively at positions 542, 568 and 569, are replaced by His, His and Leu; a variant in which Gly and Ser, respectively at positions 282 and 283, are replaced by Ala and Leu; and a variant in which Ala, Phe and Pro, respectively at positions 450, 537 and 545, are replaced by Pro, Leu and Ile; andwherein said variant of the CryIAc insecticidal fragment is selected from the group consisting of: Phe at position 148 being replaced by Leu; Leu at position 366 being replaced by Phe; Phe at position 440 being deleted and Asn at position 442 being replaced by Ser.
 4. The method according to claim 1, wherein said Ostrinia nubilalis is further contacted with a protein selected from the group consisting of:a CryIAb protein comprising the amino acid sequence of SEQ ID No. 2 or a variant thereof having at least one mutation, wherein said at least one mutation comprises Asp at position 542 being replaced by His; Thr at position at position 568 being replaced by His; Val at position 569 being replaced by Leu; Gly at position 282 being replaced by Ala; Ser at position 283 being replaced by Leu; Ala at position 450 being replaced by Pro; Phe at position 537 being replaced by Leu; and Pro at position being replaced by Ile; and a CryIAc protein comprising the amino acid sequence of SEQ ID NO. 3 or a variant thereof having at least one mutation selected from the group consisting of Phe at position 148 being replaced by Leu; Leu at position 366 being replaced by Phe; Phe at position 440 being deleted; and Asn at position 442 being replaced by Ser.
 5. The method according to claim 1, wherein said contacting step is carried out with a microorganism expressing said protein, insecticidal fragment, or variants thereof.
 6. The method according to claim 2, wherein said contacting step is carried out with a microorganism expressing said proteins, insecticidal fragments, or variants thereof.
 7. The method according to claim 1, wherein said contacting step is carried out with a plant stably transformed with a DNA sequence encoding said protein, insecticidal fragment or variants thereof.
 8. The method according to claim 1, wherein said contacting step comprises growing or planting corn stably transformed with a DNA sequence encoding said protein, insecticidal fragment or variants thereof in a field infestable by Ostrinia nubilalis.
 9. The method according to claim 2, wherein said contacting step comprises growing or planting corn stably transformed with a group of DNA sequences encoding said proteins, insecticidal fragments or variants thereof in a field infestable by Ostrinia nubilalis.
 10. A method for producing corn protected against Ostrinia nubilalis, comprising:(a) transforming a corn cell with a DNA sequence encoding said protein, insecticidal fragment or variants thereof according to claim 1; (b) regenerating a transformed corn plant from said cell; and (c) planting said transformed corn plant in a field infestable by Ostrinia nubilalis.
 11. A method for producing corn protected against Ostrinia nubilalis, comprising:(a) transforming a corn cell with a group of DNA sequences encoding said proteins, insecticidal fragments or variants thereof according to claim 3; (b) regenerating a transformed corn plant from said cell; and (c) planting said transformed corn plant in a field infestable by Ostrinia nubilalis.
 12. The method according to claim 10, which further comprises the steps of: obtaining progeny plants producing said protein, insecticidal fragment or variants thereof from said transformed corn plant and growing said progeny plants in a field infestable by Ostrinia nubilalis.
 13. The method according to claim 11, which further comprises the steps of: obtaining progeny plants producing said proteins, insecticidal fragments or variants thereof from said transformed corn plant and growing progeny plants in a field infestable by Ostrinia nubilalis.
 14. A method for combatting or controlling Ostrinia nubilalis, comprising growing corn plants expressing said protein, insecticidal fragment or variants thereof according to claim 1 in a field infestable by Ostrinia nubilalis.
 15. The method according to claim 1, wherein said insecticidal fragment or variants thereof are about 66 kD.
 16. The method according to claim 4, wherein said variant of the CryIAb protein is selected from the group consisting of: a variant in which Asp, Thr and Val, respectively at positions 542, 568 and 569 are replaced by His, His and Leu; a variant in which Gly and Ser, respectively at positions 282 and 283 are replaced by Ala and Leu; and a variant in which Ala, Phe and Pro, respectively at positions 450, 537 and are replaced by Pro, Leu and Ile; andwherein said variant of the CryIAc protein is selected from the group consisting of: Phe at position 148 being replaced by Leu; Leu at position 366 being replaced by Phe; Phe at position 440 being deleted; and Asn at position 442 being replaced by Ser.
 17. The method according to claim 6, wherein said microorganism is a plant-associated microorganism.
 18. The method according to claim 3, wherein said contacting step is carried out with a plant, stably transformed with at least one DNA sequence or group of DNA sequences encoding said proteins, insecticidal fragments or variants thereof.
 19. The method according to claim 16, wherein said contacting step is carried out with a plant, stably transformed with at least one DNA sequence or group of DNA sequences encoding said proteins, insecticidal fragments or variants thereof.
 20. The method according to claim 3; wherein said contacting step comprises growing or planting corn stably transformed with a DNA sequence encoding said proteins, insecticidal fragments or variants thereof in a field infestable by Ostrinia nubilalis.
 21. The method according to claim 16, wherein said contacting step comprises growing or planting corn stably transformed with a DNA sequence encoding said proteins, insecticidal fragments or variants thereof in a field infestable by Ostrinia nubilalis.
 22. The method according to claim 5, wherein said microorganism is a plant-associated microorganism.
 23. The method according to claim 7, wherein said plant is a cereal plant.
 24. The method according to claim 23, wherein said cereal plant is corn. 